The goal of mitosis is to achieve perfect chromosome segregation, with two resulting daughter cells containing exactly the same genetic material. Successful division of genetic material is critical to human health, since defects in chromosome segregation have been implicated in the formation of birth defects and in the initiation and progression of human tumors. Integral to the process of chromosome segregation is the formation and regulation of kinetochore-microtubule (MT) attachments. The kinetochore is a large protein assemblage residing atop centromeric DNA on mitotic chromosomes. This structure serves as the attachment point between chromosomes and spindle MTs where force is generated to power chromosome movements to the spindle equator in metaphase and subsequently to the opposing spindle poles during anaphase. Work from many labs has demonstrated that the NDC80 complex, made of four proteins (Hec1/Ndc80, Nuf2, Spc24, and Spc25), serves as the primary attachment point between kinetochores and MTs. Although this is an intensive area of study, how this complex generates MT binding sites in cells remains unclear. In addition to generating robust attachments to MTs, kinetochores must also precisely regulate the strength of these attachments throughout mitosis. In early mitosis, kinetochore-MT turnover must be high so that incorrectly attached MTs can be released. As mitosis progresses, however, correctly attached MTs must be stabilized so that forces can be generated for chromosome movement and to silence the spindle assembly checkpoint (a mechanism the cell uses to ensure that all chromosomes are properly attached and aligned before exiting mitosis). The essential kinase Aurora B has been implicated in regulating kinetochore-MT attachment strength during mitosis through phosphorylation of outer kinetochore proteins. One key substrate is Hec1, a member of the NDC80 complex. Aurora B has been demonstrated to phosphorylate multiple sites within the unstructured tail region of Hec1, and this phosphorylation results in weakened kinetochore-MT attachments in vivo and decreased NDC80-MT affinity in vitro. Although it is clear that phosphorylation of the tail domain affects formation and regulation of kinetochore-MT attachments, how it does so remains an area of active study. In this proposal, I will carry out experiments to further our understanding of how th kinetochore-MT attachments are generated and regulated in vertebrate cells and how attachment status is signaled to the spindle assembly checkpoint machinery. Specifically, I will use a silence and rescue approach to investigate how the number and position of Aurora B kinase phosphorylation events affect kinetochore-MT attachment dynamics during mitosis. Additionally, I will use non-phosphorylatable Hec1 mutants to test whether the spindle assembly checkpoint responds to MT attachment status, tension across sister kinetochores, or tension within individual kinetochores. Together, these experiments should have a significant impact of our understanding of mitotic regulation in vertebrate cells.